CN116102626A - Nucleic acid probe and preparation method and application thereof - Google Patents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
- C07K14/003—Peptide-nucleic acids (PNAs)
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
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Abstract
The invention discloses a nucleic acid probe, a preparation method and application thereof, comprising the following steps: step S1, preparing PNA with activity; step S2, coupling the active AuNPs solution to the cysteine-activated PNA to complete the preparation. By the method, the PNA has obviously improved water solubility, improved hybridization affinity and realized PNA pharmaceutical function. The PNA is used for replacing the oligonucleotide to prepare the test strip, so that the test strip is more stable and better in specificity than the common test strip. PNA is more stable as a probe than ordinary nucleic acid probes, is not affected by environmental factors, and has a stronger binding ability to RNA than RNA and ordinary oligonucleotides. The detection of the result is facilitated.
Description
Technical Field
The invention belongs to the technical field of chemical synthesis, and particularly relates to a nucleic acid probe, a preparation method and application thereof.
Background
Probe technology, chemical and biological technology for detection analysis using chemical substances (probes) that specifically recognize or label target molecules and are suitable for direct detection. The probe belongs to a high-efficiency analysis reagent and has the greatest characteristics of strong specificity and high sensitivity. The probe technology plays a great role in biological macromolecule research, drug analysis, judicial identification, clinical disease diagnosis and treatment and the like. Nucleic acid probes based on the principle of hybridization of nucleic acid molecules are the most common type of probes. Nucleic acid probes detect specific nucleic acid sequences using the hybridization properties of nucleic acid molecules. From the beginning of genetic engineering technology, nucleic acid probes are used in many fields such as gene research, SNP detection, STR detection, tumor research, etc.
Peptide Nucleic Acid (PNA), which is a kind of DNA analogue with polypeptide skeleton to replace sugar phosphate main chain, is a new kind of DNA analogue, i.e. with neutral peptide chain amide 2-amino ethyl glycine bond to replace pentose phosphate diester bond skeleton in DNA, and the rest is identical to DNA, PNA can recognize and combine DNA or RNA sequence in Watson-Crick base pairing mode to form stable double helix structure. Because peptide nucleic acid is not negatively charged and does not have electrostatic repulsion with DNA and RNA, the stability and specificity of the combination are greatly improved; unlike hybridization between DNA and RNA or between DNA and RNA, hybridization between PNA and DNA or RNA is hardly affected by the salt concentration of the hybridization system, and hybridization ability with DNA or RNA molecule is far better than that of binding between DNA and RNA or between DNA and RNA, and its superiority is represented by high hybridization stability, excellent specific sequence recognition ability and no hydrolysis by nuclease and protease. And may be co-transfected into cells in conjunction with a ligand. These are all advantages over other oligonucleotides. These characteristics, coupled with low toxicity and immunogenicity, make PNA one of the most effective tools in biomedical applications, both diagnostic and therapeutic. A number of modified PNAs have been synthesized by modification of the backbone, the bases, and the manner and positions of the backbone and base linkages. These novel PNAs have significantly improved water solubility, improved hybridization affinity, etc., and have achieved the pharmaceutical functions and objectives of the PNA of researchers.
Nano gold (AuNPs) refers to gold tiny particles, and gold nanoparticles with different shapes correspond to different application purposes. By the day, people have made gold nanoparticles with various shapes, mainly cassia-shaped, spherical, charged, flower-shaped, polyhedral, star-shaped and the like, and the gold nanoparticles with different shapes have unique advantages. The particle size is 1-100 nm, has high electron density, dielectric property and catalysis, can be combined with various biological macromolecules, and does not influence the biological activity. AuNPs have been new materials developed and applied in the aspects of catalysis, detection, biosensing, drug carriers, biological probes, gene chips and the like in the 21 st century because of their good optical, electrical and biological affinity and other properties.
Currently, the use of modified PNAs as a tool for molecular biology in gene diagnosis and detection, gene chips, biosensors, etc. has been gradually matured. Can be widely used for molecular hybridization, in situ hybridization, mutation analysis, anticancer and antiviral antisense nucleic acid research and application of pathogen and genetic disease detection.
Immobilization of the biological probe on the transducer may be one of the most critical steps in biosensor manufacturing. In fact, efficient and reproducible immobilization, while ensuring stable binding to the sensor surface throughout the analysis process, is the most critical part of the biosensor manufacturing process. The substitution of peptide nucleic acid for the substituted oligonucleotide is used for the preparation of hybridization probes, and has the advantages not available in other oligonucleotides.
Disclosure of Invention
The invention aims to provide a nucleic acid probe, a preparation method and application thereof, which are used for solving the problems that in the prior art, oligonucleotides are adopted and have no hybridization stability and specific sequence recognition capability.
In order to solve the technical problems, the invention adopts the following technical scheme:
a nucleic acid probe comprising the steps of:
step S1, preparing PNA with activity;
step S2, coupling the AuNPs solution with activity to the PNA activated by cysteine to complete the preparation;
in step S1, the preparation of active PNA is specifically: PNA has the formula:
a cysteine was added to the 5-terminus of the PNA sequence to render PNA active;
the PNA sequence is specifically: GCAACTTCTATGTAT;
the active PNA sequences are: cys-GCAACTTCTATGTAT.
A preparation method of a nucleic acid probe comprises the following specific steps of coupling AuNPs solution to PNA:
step A1, firstly, selecting an AuNPs solution with the particle size of 15-20nm, and centrifuging the AuNPs solution at a low temperature;
step A2, removing part of supernatant after centrifugation, and performing secondary concentration; and adjusting the Ph value of the concentrated AuNPs solution to 7.0-7.2;
step A3, adding 20 mu L of PNA with the concentration of 10 mu mol/L into the concentrated and resuspended 100 mu LAuNPs solution, and placing the solution at the temperature of 4 ℃ in a dark place for 24 hours;
step A4, adding 100 mu L of mixed solution containing 40 mmol/L phosphate buffer solution and 0.8 mol/LNaCl into the AuNPs-PNA solution after being placed for 24 hours in a dark place;
step A5, placing the AuNPs-PNA solution mixed with the mixed solution at 4 ℃ for 48 hours in a dark place, centrifuging at 10000r/min for 20 min at 4 ℃, and removing the supernatant after centrifugation;
step A6, re-suspending and precipitating the centrifuged AuNPs-PNA solution by using 500 mu L of mixed solution containing 20mmol/L phosphate buffer solution and 0.6mol/L NaCl;
step A7, centrifuging the AuNPs-PNA solution subjected to heavy suspension precipitation at 4 ℃ for 10 min at 10000r/min, and removing supernatant after centrifugation;
and (A8) finally, re-suspending and precipitating the centrifuged AuNPs-PNA solution by using 200 mu L of mixed solution containing 20mmol/L phosphate buffer solution and 0.6 mol/LNaCl, and preserving at 4 ℃ in a dark place to finish coupling of the AuNPs and the PNA.
Preferably, the nucleic acid probe is used for detection of chemical substances.
Compared with the prior art, the invention has the following beneficial effects:
by the method, the PNA has obviously improved water solubility, improved hybridization affinity and realized PNA pharmaceutical function. The PNA is used for replacing the oligonucleotide to prepare the test strip, so that the test strip is more stable and better in specificity than the common test strip. PNA is more stable as a probe than ordinary nucleic acid probes, is not affected by environmental factors, and has a stronger binding ability to RNA than RNA and ordinary oligonucleotides. The detection of the result is facilitated.
Drawings
FIG. 1 is a diagram showing the ultraviolet visible spectrum of an AuNPs-PNA probe of the present invention;
FIG. 2 is a graph of AuNPs solutions of this invention at different pH values;
FIG. 3 is a graph showing the experimental comparison of UV and oligonucleotide nanogold probes of AuNPs-PNA probes of the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the present invention, PNA refers to peptide nucleic acid (Peptide nucleic acids, PNA); auNPs refers to nano-gold.
Example 1
As shown in fig. 1 to 3, a nucleic acid probe comprises the steps of:
step S1, preparing PNA with activity;
step S2, coupling the AuNPs solution with activity to the PNA activated by cysteine to complete the preparation;
in step S1, the preparation of active PNA is specifically: PNA has the formula:
a cysteine was added to the 5-terminus of the PNA sequence to render PNA active;
the PNA sequence is specifically: GCAACTTCTATGTAT;
the active PNA sequences are: cys-GCAACTTCTATGTAT.
A preparation method of a nucleic acid probe comprises the following specific steps of coupling AuNPs solution to PNA:
step A1, firstly, selecting an AuNPs solution with the particle size of 15-20nm, and centrifuging the AuNPs solution at a low temperature;
step A2, removing part of supernatant after centrifugation, and performing secondary concentration; and adjusting the Ph value of the concentrated AuNPs solution to 7.0-7.2;
step A3, adding 20 mu L of PNA with the concentration of 10 mu mol/L into the concentrated and resuspended 100 mu LAuNPs solution, and placing the solution at the temperature of 4 ℃ in a dark place for 24 hours;
step A4, adding 100 mu L of mixed solution containing 40 mmol/L phosphate buffer solution and 0.8 mol/LNaCl into the AuNPs-PNA solution after being placed for 24 hours in a dark place;
step A5, placing the AuNPs-PNA solution mixed with the mixed solution at 4 ℃ for 48 hours in a dark place, centrifuging at 10000r/min for 20 min at 4 ℃, and removing the supernatant after centrifugation;
step A6, re-suspending and precipitating the centrifuged AuNPs-PNA solution by using 500 mu L of mixed solution containing 20mmol/L phosphate buffer solution and 0.6mol/L NaCl;
step A7, centrifuging the AuNPs-PNA solution subjected to heavy suspension precipitation at 4 ℃ for 10 min at 10000r/min, and removing supernatant after centrifugation;
and (A8) finally, re-suspending and precipitating the centrifuged AuNPs-PNA solution by using 200 mu L of mixed solution containing 20mmol/L phosphate buffer solution and 0.6 mol/LNaCl, and preserving at 4 ℃ in a dark place to finish coupling of the AuNPs and the PNA.
Preferably, the nucleic acid probe is used for detection of chemical substances.
Further, in step A1, the temperature of the low-temperature centrifugation is 2℃to 6℃and preferably 4 ℃.
Further, in the step A2, the specific range of the supernatant after removing part of the centrifugation is: 600-800 mul.
Further, in step A2, the secondary concentration is performed by low-temperature centrifugation at a temperature of 2℃to 6℃and preferably 4 ℃.
Further, before the AuNPs were coupled with PNA, the AuNPs were washed five times in Dimethylformamide (DMF), and the silylated AuNPs reaction was performed under the following conditions:
and 2, placing the synthesized AuNPs and the prepared alkylation reaction liquid in a thermal circulator at 65 ℃ for 2 hours, accelerating the alkylation reaction, improving the connection effect of the AuNPs and the PNA, and enabling the connection effect of the AuNPs and the PNA to be better.
FIG. 1 is a UV-visible spectrum of an AuNPs-PNA probe, showing a red shift in wavelength after successful attachment of AuNPs and PNA, with a maximum absorption peak rising from 520nm to 540nm, demonstrating successful preparation of the AuNPs-PNA probe.
Example two
This example provides a process for preparing AuNPs solution coupled to PNA. In order to study the optimal pH conditions for the thiol-modified PNA to connect with AuNPs, the pH values of the gold nanosolutions are respectively adjusted to 6.6, 6.8, 7.0, 7.2 and 7.4, 50 mu L of 10 mu mol/LPNA solution is then respectively added, the mixture is fully and uniformly mixed, and the mixture is kept stand at 4 ℃ for 24 hours, so that the final reaction condition is observed.
When the pH of the solution does not reach the optimal pH of PNA and AuNPs, the salt ion effect of NaCl in the reaction solution can neutralize the negative charge on the surface of the AuNPs in an unbound state, so that the AuNPs are aggregated and sedimentation occurs, and the color of the solution is changed from red to blue. At the optimum pH, the solution remained reddish and did not precipitate.
The results are shown in FIG. 2: at pH 6.6, the solution was pale blue, close to five with a small amount of precipitation; at pH 6.8, the solution appeared bluish gray with concomitant precipitation; when the pH is 7.0, the solution is reddish in wine, but a small amount of sediment is separated out; when the pH is 7.2, the solution is wine red and has no precipitation; when the pH was 7.4. The solution turned dark purple in color and a small amount of precipitation occurred, so that the optimal pH for AuNPs-PNA probe ligation was 7.2. Coupling can occur at a pH in the range of 7.0-7.2 and peracids or overbases can deactivate the nanogold.
The pH of the AuNPs solution was adjusted to the optimum pH. Then taking 6 separation tubes, respectively adding 100 mu L of AuNPs solution, respectively adding 1, 5, 10, 20, 30 and 40 mu L of 10 mu mol/L activated PNA solution into each tube, uniformly mixing, standing for 24 hours, adding 0.6mol/L NaCI and 20mmol/L phosphate buffer (pH 7.0) with the same volume as the peptide nucleic acid solution into each tube, fully uniformly mixing, standing for 48 hours at 4 ℃, observing the color, precipitation and other changes of the AuNPs solution of each tube, and determining the optimal PNA probe quantity for modifying the AuNPs to be 1:20.
Example III
As shown in FIG. 3, the oligonucleotide nano gold probe and the peptide nucleic acid nano probe are coated on the same rapid detection test strip, the No. 1 and the No. 3 oligonucleotide probes, the No. 2 AuNPs-PNA probes are used for capturing target RNA with the same concentration, so that the No. 2 AuNPs-PNA probes have stronger capturing capability and more stable color development.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (3)
1. A nucleic acid probe, characterized in that: the method comprises the following steps:
step S1, preparing PNA with activity;
step S2, coupling the AuNPs solution with activity to the PNA activated by cysteine to complete the preparation;
in step S1, the preparation of active PNA is specifically: PNA has the formula:
a cysteine was added to the 5-terminus of the PNA sequence to render PNA active;
the PNA sequence is specifically: GCAACTTCTATGTAT;
the active PNA sequences are: cys-GCAACTTCTATGTAT.
2. A method for preparing a nucleic acid probe is characterized in that: the specific steps for coupling the AuNPs solution to PNA are:
step A1, firstly, selecting an AuNPs solution with the particle size of 15-20nm, and centrifuging the AuNPs solution at a low temperature;
step A2, removing part of supernatant after centrifugation, and performing secondary concentration; and adjusting the Ph value of the concentrated AuNPs solution to 7.0-7.2;
step A3, adding 20 mu L of PNA with the concentration of 10 mu mol/L into the concentrated and resuspended 100 mu LAuNPs solution, and placing the solution at the temperature of 4 ℃ in a dark place for 24 hours;
step A4, adding 100 mu L of mixed solution containing 40 mmol/L phosphate buffer solution and 0.8 mol/LNaCl into the AuNPs-PNA solution after being placed for 24 hours in a dark place;
step A5, placing the AuNPs-PNA solution mixed with the mixed solution at 4 ℃ for 48 hours in a dark place, centrifuging at 10000r/min for 20 min at 4 ℃, and removing the supernatant after centrifugation;
step A6, re-suspending and precipitating the centrifuged AuNPs-PNA solution by using 500 mu L of mixed solution containing 20mmol/L phosphate buffer solution and 0.6mol/L NaCl;
step A7, centrifuging the AuNPs-PNA solution subjected to heavy suspension precipitation at 4 ℃ for 10 min at 10000r/min, and removing supernatant after centrifugation;
and (A8) finally, re-suspending and precipitating the centrifuged AuNPs-PNA solution by using 200 mu L of mixed solution containing 20mmol/L phosphate buffer solution and 0.6 mol/LNaCl, and preserving at 4 ℃ in a dark place to finish coupling of the AuNPs and the PNA.
3. Use of a nucleic acid probe, characterized in that: the nucleic acid probe prepared in claim 1 for detection of chemical substances.
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